1. Field of the Invention
This invention primarily relates to a method to prevent or inhibit cell adhesion and/or biofilm formation by a microorganism, in which use of 1,2,3,4,6-penta-O-galloyl-D-glucopyranose (PGG) is involved therein.
2. Description of the Related Art
Nearly 99% of the microorganisms living in this planet thrive in microbial communities known as biofilms. They are formed by adhesion of cells to surfaces through an exopolymeric matrix. This matrix is important both in the formation and structure of the biofilm, and also to the protection of the cells since it may prevent the access of antimicrobials and xenobiotics to the cells inside the biofilm and confer protection against environmental stresses such as UV radiation, pH shifts, osmotic shock and desiccation. Fossilised biofilms of up to 3.5 billion years are among the oldest records of life on Earth. Biofilms are the planet's most successful form of colonialism as they live on soils, sediments, mineral, plant and animal surfaces, even under extreme environments, from glaciers to hot vents. Biofilms are even able to thrive in highly irradiated areas of nuclear power plants.
Cell aggregation is advantageous and required in several processes, namely in biological wastewater treatment and bioremediation systems. However, they constitute a serious problem in many industrial processes (e.g., paper, food, cosmetic and pharmaceutical industries) because they can cause corrosion and limit mass and heat transfer in pipes and tubes and mainly in water distribution systems and healthcare environments as they are a source of microbial infections. Furthermore, biofilms can also bioaccumulate metals and toxic compounds. Biofilms can contaminate contact lenses, catheters, endotracheal tubes, mechanical cardiac valves, prosthetic joints, surgical sutures, etc. Biofilms can result in surgical site infections, and cells in biofilms have higher resistance to antibiotics and biocides than planktonic cells and gene transfer is possible horizontally, which improves the exchange of genes between resistant and non-resistant strains.
Bacterial strains that do not produce exopolymeric substances (EPS) present lower adherent abilities than slime-producing strains. EPS is particularly valuable after the initial phase of adhesion in organisms, conferring protection against phagocytosis, interference with the cellular immune response and reduction of antibiotic potency. In fact, the host immune system is, in general, capable of rapidly killing non-adherent bacteria. The slow growth rate observed in biofilms and/or transport limitations of nutrients, metabolites and oxygen between the surface and the interior of the biofilm could be responsible for an increased antibiotic resistance over planktonic cells. Furthermore, the EPS matrix acts as an anchor, securing the cells and preventing their detachment under flow conditions, although detachment of cells may also be an important process for propagation and formation of new colonies.
The above is an excerpt from a review article, namely Carla C. C. R. de Carvalho (2007), Recent Patents on Biotechnology, 1, 49-57, which provides a thorough discussion of a number of scientific literatures and patent documents in relation to the prevention and control of biofilm proliferation in medical devices and water supply systems, as well as the development on anti-microbial surfaces, detergents and biocides. This review article and the references referred to therein are incorporated herein by reference in their entirety.
Biofilms can colonize on almost all surfaces, from glass to steel, and from cellulose to silicone, which are the main materials used to produce medical devices. Bacterial biofilms are frequently found in clinical and medical environments, and according to statistics, 65 percent of bacterial infections are associated with biofilms formed by bacteria. Many clinically important pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococci, etc., have the ability to form biofilm. Inasmuch as bacteria living in biofilm have higher resistance to the attack of the host immune system and the toxic effect of antibiotics, it becomes difficult to treat infections of this kind. In particular, it is extremely hard to eradicate a bacterial biofilm once formed in an implanted medical device such as a cardiac catheter, a urethral catheter, a prosthetic joint, an artificial heart valve, an artificial blood vessel, etc., and replacing the biofilm-contaminated device with a new one is imperative, which leads to a year-by-year rise in medical expenses.
Amongst pathogens that cause nosocomial infections, Staphylococcus aureus is of great concern. Staphylococcus aureus forms biofilms on medical devices and causes pneumonia, meningitis, endocarditis, osteomyelitis and septicemia (Friedrich Götz (2002), Mol. Microbiol., 43 (6):1367-1378). Formation of biofilm by S. aureus is closely associated with the synthesis of an extracellular polysaccharide substance (EPS), namely polysaccharide intercellular adhesion (PIA), which is a β-1,6-linked N-acetyl (succinyl) glucosamine polymer synthesized by enzymes encoded by an ica operon (Sarah E. Cramton et al. (1999), Infect. Immun., 67 (10):5427-5433). In addition, the proteins of S. aureus that contribute to biofilm formation include fibronectin-binding proteins A and B (Eoghan O'Neill et al. (2009), J. Med. Microbiol., 58:399-402), collagen-binding protein (Patrick Ymele-Leki and Julia M. Ross (2007), Appl. Environ. Microbiol., 73 (6):1834-1841), SasG surface protein (Rebecca M. Corrigan et al. (2007), Microbiology, 153:2435-2446), and the biofilm associated protein bap (M. Ángeles Tormo et al. (2005), Microbiology, 151:2465-2475). Meanwhile, the pertinacious form of biofilm formation is inhibited by extracellular proteases produced by the organism (Elena Stary et al. (2010), Appl. Environ. Microbiol., 76 (3):680-687). These reports illustrate that factors on the bacterial surface facilitate attachment of bacteria and establishment of multilayered cell clusters on a solid surface, which are crucial to biofilm formation (Friedrich Götz (2002), supra). Owing to high resistance to antibiotics of biofilm-imbedded staphylococci (Kim Lewis (2001), Antimicrob, Agents Chemother., 45 (4):999-1007), biofilm-associated infections are extremely difficult to treat, necessitating the development of drugs that prevent and destroy biofilm.
In the last two decades, numerous investigators specializing in the research and development of anti-biofilm drugs and anti-biofilm medical instruments have identified several substances useful for preventing the formation or removal of staphylococcal biofilms. For example, lysostaphin, which is a peptidoglycan-degrading enzyme, prevents staphylococcal biofilm formation (Julie A. Wu et al. (2003), Antimicrob. Agents Chemother., 47 (11):3407-3414); N-acetylcysteine (NAC), which is a clinical drug, reduces EPS production, subsequently inhibiting biofilm formation (C. Pérez-Giraldo et al. (1997), J. Antimicrob. Chemother., 39:643-646); iodoacetamide (IDA) and N-ethyl maleimide (NEM), which are inhibitors of N-acetyl-D-glucosamine-1-phosphate acetyltransferase that catalyzes the biosynthesis of UDP-N-acetylglucosamine, i.e., a precursor of PIA, also inhibit biofilm formation (Euan Burton et al. (2006), Antimicrob. Agents Chemother., 50:1835-1840); and dispersin B, which hydrolyzes β-1,6-N-acetyl-glucosamine, disperses mature biofilm and is an agent potentially useful in anti-biofilm therapy (Yoshikane Itoh et al. (2005), J. Bacteriol., 187 (1):382-7). In addition, there have been researches investigating the use of different clinical antibiotics in combination to inhibit bacterial biofilm formation (Euan Burton et al. (2006), supra; and Saima Aslam et al. (2007), Antimicrob. Agents Chemother., 51 (4):1556-8).
While the target drugs or candidate drugs studied in the earlier reports are considered to have potential as an anti-biofilm agent (Saima Aslam et al. (2007), supra; Nicholas Beckloff et al. (2007), Antimicrob. Agents Chemother., 51 (11):4125-32; Ross P. Carlson et al. (2008), J. Biomater. Sci. Polymer Ed., 19 (8): 1035-1046; I. Raad et al. (2008), J. Antimicrob. Chemother., 62:746-650; Prabha Ramritu et al. (2008), Am. J. Infect. Control, 36 (2) 104-117; and Guo-Xian Wei et al. (2006), J. Antimicrob. Chemother., 57:1100-9), the greatest concern for enzymatic drugs to be used in clinic is the extremely high cost thereof, and drugs exhibiting bacterial-killing effect(s) are liable to induce resistant strains. Furthermore, the clinical efficacies of these target drugs or candidate drugs as studied in the above reports have yet to be verified.
There also exist numerous published patent documents disclosing a diversity of approaches to inhibit or prevent biofilm formation on various surfaces, including, e.g., WO 2008/132718 A2, WO 2010/072257 A1, WO 2010/112848 A2, WO 2010/144686 A1, U.S. Pat. No. 7,691,418 B2, US 20100152101 A1 (corresponding to TW200913996A), US 20090192192 A1, US 20090255536 A1, US 20090304621 A1, US 20100087769 A1, US 20100285084 A1, US 20100298208 A1, US 20110008402 A1, US 20110076312 A1, US 201100763332 A1, and US 20110086101 A1. These patent documents are incorporated herein by reference in their entirety.
In order to exploit anti-biofilm agents that are effective at low concentrations, non-toxic and biodegradable, health and environment friendly, and cost-saving, the applicants screened forty eight compounds isolated from plants commonly used in Chinese medicine, and surprisingly found that 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (β-PGG), either in a solution or coated on solid surfaces, did not inhibit the growth of tested microorganisms, yet prevented biofilm formation on solid surfaces. The α-anomer of β-PGG was also found to exhibit similar anti-biofilm effect. It is contemplated that PGG, either in the β-form or the α-form or a mixture of the α- and β-forms, may act as a potent anti-biofilm agent.